The present invention relates, in general, to immunosuppression and, in particular, to a method of inducing an immunosuppressive effect. The invention further relates to a method of screening compounds for immunosuppressive activity.
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes and postmeiotic male germ cells. It is present in the nucleus of cells in which it is expressed (Jensen et al, Proc. Natl. Acad. Sci. USA 88:2850 (1991)). CaMKIV phosphorylates and activates the cyclic AMP response element binding proteins CREB and CREMxcfx84 in a manner analogous to protein kinase A (Matthews et al, Mol. Cell. Biol. 14:6107 (1994); Sun et al, Genes Dev. 8:2527 (1994); Enslen et al, J. Biol. Chem. 269:15320 (1994)).
In the absence of Ca2+/calmodulin, CaMKIV is inactive. Activation requires three events: i) binding of Ca2+/calmodulin; ii) phosphorylation of a single threonine residue present in the activation loop by a separate protein kinase that is also Ca2+/calmodulin-dependent; and iii) autophosphorylation of serine residues present in the extreme N-terminus that is required to relieve a novel form of autoinhibition.
The gene for rat CaMKIV has been cloned and found to span 42 kb of DNA. The gene encodes 3 proteins namely, the xcex1 and xcex2 forms of CaMKIV that differ only in that the xcex2 form contains a 28 amino acid N-terminal extension as well as calspermin (Sun et al, J. Biol. Chem. 270:29507 (1995)). Calspermin is the C-terminal 169 amino acids of CaMKIV that binds Ca2+/calmodulin and is expressed only in posmeiotic male germ cells. The promoter for calspermin resides in the penultimate intron of the CaMKIV gene and is regulated by two CREs. Available data suggest that rearrangement of chromatin during meiosis together with the expression of CREMxcfx84 at high levels are sufficient to control expression of the calspermin promoter during spermatogenesis. On the other hand, the developmental expression of CaMKIV in cells of the brain and T cells appears to be controlled by thyroid hormone mediated via the thyroid hormone receptor xcex1.
Prior to the present invention, the specific role of CaMKIV in T cell activation had not been defined and the potential importance of CaMKIV as a target for immunosuppressive agents had not been suggested. Indeed, currently available immunosuppressive drugs, as well as new generation drugs presently under development, target calcineurin. Since calcineurin is present in all cells, not just T cells, these drugs are associated with a plethora of side effects. The present invention provides a method of identifying immunosuppressive agents that target CaMKIV and that are T cell specific and thus substantially free of adverse side effects.
It is one object of the invention to provide a method of inducing an immunosuppressive effect, while avoiding undesirable side effects.
It is another object of the invention to provide a method of screening compounds for immunosuppressive activity.
It is a further object of the invention to provide a method of inhibiting CREB phosphorylation specifically in T cells.
The foregoing objects are met by the present invention which results, at least in part, from the recognition that inhibition of CaMKIV prevents phosphorylation of CREB and thereby activation of T cells.
Further objects and advantages of the present invention will be clear from the description that follows.